Changes in Protein Kinase C during Vitellogenesis in the Crayfish ...

General and Comparative Endocrinology 118, 200 –208 (2000) doi:10.1006/gcen.2000.7471, available online at http://www.idealibrary.com on

Changes in Protein Kinase C during Vitellogenesis in the Crayfish Cherax quadricarinatus—Possible Activation by Methyl Farnesoate Yoram Soroka,* Amir Sagi,† Isam Khalaila,† Uri Abdu,† and Yoram Milner* ,1 *Department of Biological Chemistry, Life Science Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and †Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Accepted February 4, 2000

and methyl farnesoate stimulated activation of PKC ␣ in organ culture, causing its translocation from the cytosol to the membranes and inducing autophosphorylation of threonine residues. The changes in PKC isoenzymes during ovarian maturation in the crayfish suggest their involvement in this process as well as a possible regulatory role for methyl farnesoate through a direct effect on some PKC isoenzymes. © 2000 Academic Press Key Words: protein kinase C; ovary; vitellogenesis; Crustacea; Decapoda; crayfish; Cherax quadricarinatus; methyl farnesoate.

During ovarian maturation in the crayfish Cherax quadricarinatus, changes in ovarian protein kinase C (PKC) isoenzymes take place in parallel to yolk accumulation (as shown by immunoblot analysis). Significant changes were recorded in the amounts of specific isoenzymes and in their distribution between the cytosol and the membranes. Ovarian maturation was accompanied by the appearance of high- and low-molecular-weight immunoreactive PKC isoenzyme species. Among the isoenzymes tested, PKC ␣ was the most clearly activated during ovarian maturation, as shown by significant translocation from the cytosol to the particulate fraction and the appearance of high-molecular-weight species. Moreover, a similar picture was obtained in the ovaries of intersex individuals upon induction of secondary vitellogenesis by androgenic gland ablation. Immunohistological staining showed PKC ␣ to be localized mainly in the cytosol of premature oocytes, whereas in later maturation stages, it was concentrated around the nucleus in a vesicular structure and in the oocyte membrane. In secondary vitellogenic stages, PKC was localized in the plasma membrane and apparently in follicular cells. In addition, its activity was demonstrated by in vitro phosphorylation assays of a crayfish ovarian homogenate. Activation of total PKC phosphorylation of histone, an external substrate, was induced by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate (TPA) or methyl farnesoate. Both TPA

In higher eukaryotes, protein kinase C (PKC) enzymes constitute an integral part of the signal transduction chain in many biological processes, such as control of cell proliferation, differentiation, and maturation (Nishizuka, 1986). In mammals, for example, there are at least 12 PKC isoenzymes (Dekker and Parker, 1994; Hofmann, 1997) which are classified into three main subgroups (Knopf et al., 1986)— classic, novel, and atypical. The classic (c-PKC) group, ␣-, ␤I-, ␤II-, and ␥-PKCs, are Ca 2⫹-dependent enzymes with a Ca 2⫹-binding site (C 2 domain). The novel (n-PKC) Ca 2⫹-insensitive PKCs, ␦-, ⑀-, ␪-, ␩-, and ␮-PKCs, lack the C 2 sequence. Both groups have two cystein-rich C 1 domains that bind diacylglycerol and phorbol esters. The atypical isoenzymes (a-PKC), i.e., ␨-, ␭-, and ␶-PKCs, lack the C 2 domain and one of the C 1 domains. Consequently, these isoenzymes are Ca 2⫹ in-

1 To whom correspondence should be addressed. Fax: (972)-26585428. E-mail: [email protected].

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PKC and Vitellogenesis in Crayfish

sensitive and are partially activated by diacylglycerol or phorbol esters. In crustaceans, protein kinases have recently been shown to be involved in a number of systems: for example, in the mediation of photoreceptor activity in Leptograpsus variegatus (Blest et al., 1994), in synaptic transmission at neuromuscular junctions in Macrobrachium rosenbergii (Gilat and Hochner, 1990), and in the regulation of hormone release in Carcinus maenas (Baghdassarian et al., 1996). PKC ␦ has been purified from the hepatopancreas of the shrimp Penaeus monodon (Huang and Chuang, 1995). In the crayfish Cherax quadricarinatus, ovarian maturation is characterized by rapid yolk deposition (vitellogenesis) in the oocyte and fast increase in oocyte diameter and ovarian size (Sagi et al., 1996b). The main component accumulated in the oocyte during this process is vitellin, a glycolipocarotenoprotein that later serves as a source of nutrition for the developing embryo (Adiyodi and Subramonian, 1983; Quackenbush, 1991). The hormonal regulation of this process has not, however, been completely elucidated. In other arthropods, such as insects, juvenile hormones regulate the vitellogenic process in the adult female (Koeppe et al., 1985). Indications of involvement of PKC in vitellogenesis were shown by Sevala and Davey (1989, 1993). Juvenile hormone caused two different effects on a membrane preparation of vitellogenic follicular cells of Rhodnius prolixus: (a) significant increase in the phosphorylation of a membranal polypeptide in the follicular cells and (b) induction of the appearance of large spaces between the follicle cells through which vitellogenin gains access to the oocyte surface. Insect juvenile hormone III is almost identical in structure to methyl farnesoate, a terpenoid synthesized in the crustacean mandibular organ and secreted into the hemolymph (Laufer et al., 1987). Although the role and mode of action of methyl farnesoate have not been completely elucidated—and are probably not universal to all crustaceans (Homola and Chang, 1997)—methyl farnesoate does have some involvement in the crustacean reproductive system. In Libinia emarginata, for example, the levels of methyl farnesoate synthesis by mandibular organs in culture changed in relation to the stage of reproduction (Laufer et al., 1987). The compound has also been suggested to affect reproduction in the shrimp Penaeus vannamei and in the crayfish Procambarus clarkii

(Laufer, 1992; Laufer et al., 1998). In addition, methyl farnesoate was found to stimulate general protein synthesis in the ovary of M. rosenbergii during vitellogenesis (Soroka et al., 1993). A possible relationship between methyl farnesoate and PKC in the barnacle Balanus amphitrite was recently reported by Yamamoto et al. (1997), who demonstrated that methyl farnesoate induced larval metamorphosis via PKC activation. In the light of the possible involvement of PKC and methyl farnesoate in crustacean reproduction, the present study investigates the presence and changes in distribution of PKC during ovarian maturation in the crayfish C. quadricarinatus and identifies the specific isoenzymes that are activated during this process. The effects of methyl farnesoate on these PKC isoenzymes were also examined.

MATERIALS AND METHODS Animals Adult C. quadricarinatus crayfish (approximately 9 months old) were harvested from the earthen ponds of the Ministry of Agriculture, Aquaculture Research Station, Dor, Israel. Younger, early vitellogenic, females were cultured in the laboratory in 300-L tanks. The animals selected for the present study were kept in the laboratory in 100-L fresh water tanks at 28 ⫾ 2°C. The water was recirculated through a gravel biofilter. Minced fish flesh (Nile Perch), carrots, and fish pellets containing 42% protein were provided ad libitum. Animals were anesthetized in ice-cold water prior to any surgical procedure.

Organ Culture Gonads were dissected out and weighed. The gonadosomatic index (GSI) was calculated as a percentage of gonad weight to body weight. Oocyte diameter was measured under a light microscope by means of an objective micrometer (⫾0.01 mm). Mean oocyte diameter (⫾SE) for each ovary was calculated from a sample of at least 20 oocytes from the population of large oocytes (Sagi et al., 1996b). Ovaries were incubated for 1 h at 28°C with 100 nM 12-O-tetradecanoylphorbol13-acetate (TPA) in dimethyl sulfoxide (DMSO) or

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with 50 ng/ml of methyl farnesoate in ethanol or with either of the solvents alone as control (at final concentrations of 0.1%) in the following organ culture medium: Dulbecco’s modified Eagle’s medium (DMEM), pH 7.4, supplemented with 160 mg/L KCl, 240 mg/L MgCl 2 䡠 6H 2O, 3.15 g/L NaCl, and 0.5% (w/v) bovine serum albumin.

Tissue Processing, SDS–PAGE Separations, and Immunoblots Ovaries at different stages of vitellogenesis— determined according to the size of oocytes and the GSI— were homogenized on ice, with conic pestles fitted to Ependorff tubes, in 20 mM Tris buffer (pH 7.4) containing 5 mM EDTA, 10 mM EGTA, 45 mM mercaptoethanol, and a protease inhibitor mixture (1 mM benzamidine, 1 mM ⑀-amino caproic acid, 10 ␮g/ml leupeptine, 0.2 mM PMSF, and 10 ␮g/ml pepstatin). The homogenate was centrifuged (100,000g, 60 min, 4°C) to separate the cytosol from the particulate fraction. The cytosol or particulate fraction was dissolved in denaturing buffer (containing SDS and mercaptoethanol) and boiled for 5 min. Equal organ wet weight equivalents per lane (⬃2–20 ␮g protein) were loaded and separated on 8.5% SDS–PAGE (Laemmli, 1970). Rat brain cytosol was loaded on all the gels and served as a positive control for immunoreactivity with mammalian PKC. The polypeptides were electrotransferred onto nitrocellulose filters and exposed to different rabbit polyclonal anti-PKC isoenzyme antibodies (Cat. No. sc-208-sc-210, sc-212-sc-216; Santa Cruz Biotechnology, 1995) or to anti-phosphothreonine monoclonal antibody (Sigma). The filters were incubated with a secondary antibody conjugated to peroxidase and then developed by the ECL method (Pollini et al., 1993) to detect specific immunoreactive bands.

In Vitro Phosphorylation Assay Ovaries were homogenized as described above. The equivalent of 25 ␮g of ovary wet weight (approximately 0.5 ␮g protein) was transferred to a phosphorylation system (final volume 100 ␮l) containing 20 mM Tris buffer (pH 7.5), 1 mM EGTA, 4 mM MgCl 2, 30 ␮g of histone S-III, 4 mM p-nitrophenyl phosphate, 10 ␮M [␥- 32P]ATP (⬃5000 CPM/pmol), and 10 ␮g of phosphatidylserine. An homogenate aliquot was incubated


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for 20 min at 30°C in the phosphorylation system with 100 nM TPA or 50 ng of methyl farnesoate in DMSO or with DMSO alone as control (at final concentrations of 0.1%). Incorporation of 32P into histone was evaluated as follows: the substrate was absorbed on phosphocellulose paper squares (2 ⫻ 2 cm, Whatman, P-81, UK) and washed with a large excess (⬃10 ml/paper square) of 0.05 N phosphoric acid (15 min ⫻3) and H 2O (15 min ⫻2), followed by acetone and petroleum ether (10 min each). Radioactivity in the papers was measured by scintillation spectroscopy.

Immunohistological Staining Localization of PKC ␣ in the ovary was determined by fixing the tissue (⬃100 mg) for 48 h in Bouin’s solution (picric acid, formaldehyde, acetic acid, 15:5:1 v/v/v, respectively) at room temperature, followed by dehydration in steps with increasing ethanol concentrations and embedding in paraplast. Sections of 6 ␮m were cut and mounted on slides. The sections were deparaffinized in xylene and then exposed to anti-PKC ␣ antibody (Santa Cruz), 1:100, for 30 min in blocking reagent at room temperature. This procedure was followed by staining with the “Histostain Plus” kit (Zymed) using biotinylated second antibody and a horseradish peroxidase–streptavidin conjugate. Finally, diaminobenzidine (DAB) was used as a chromogenic substrate to demonstrate the presence of the antigen in the tissue.

RESULTS PKC Distribution during Ovarian Maturation The distribution of various PKC isoenzymes at different stages of vitellogenesis was analyzed in the cytosols and particulate fractions of ovarian homogenates by SDS–PAGE followed by immunoblotting. Immunoreactivity of various ovarian polypeptides with a number of anti-mammalian PKC isoenzyme antibodies was clearly evident (Fig. 1). Differences in molecular species were found among the isoenzymes: whereas most isoenzymes (␣, ␤I, ␤II, ␦, ⑀, ␪, and ␩) exhibited profound changes during the maturation

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FIG. 1. Immunoblot profile of PKC isoenzymes in the cytosol and particulate fraction of C. quadricarinatus ovaries at different vitellogenic stages. Cytosol (c) and particulate fraction (m) were separated on SDS–PAGE, transferred onto nitrocellulose, and exposed to rabbit polyclonal anti-isoenzymes of PKC. A–D represent ovaries from primary to late secondary vitellogenesis with average oocyte diameters of A ⫽ 274 ␮m, B ⫽ 575 ␮m, C ⫽ 1805 ␮m, and D ⫽ 2700 ␮m. rb, rat brain cytosol.

process, ␨ remained essentially unchanged. The most marked changes were manifested in the appearance in the particulate fraction of high- and low-molecularweight immunoreactive polypeptides, i.e., in the range of approximately 120 kDa (in ␣, ␤I, and ␪) to ⬎200 kDa (in ␦) and ⬃40 kDa (sometimes in doublets, in ␣, ␤I, ␤II, ␦, and ␩), respectively. In addition, some isoenzymes (␣, ␤I, and ␦) showed minor immunoreactive polypeptides of approx 60 kDa. Significant changes in the relative amounts of the isoenzymes ␣, ␤I, ␤II, ⑀, and ␪ were observed during vitellogenesis. For these isoenzymes, there was an increase in the total amount of various immunoreactive molecular species throughout the maturation process. Isoenzyme ␤I exhibited an increase in the relative amount of immunoreactive species in ovarian stage B (Fig. 1), followed by a decrease toward the end of the maturation process. Changes in cellular localization (translocation) of some isoenzymes were also observed. Their relative abundance in the cytosols was lower than that in the particulate fractions (Fig. 1, ␣, ␤I, ⑀). The molecular weights of the PKC immunoreactive bands of rat brain cytosol (Fig. 1, rb), which served as a positive control, were similar to those of the holoenzyme in the crayfish ovarian homogenate (75– 85 kDa).

Changes in PKC ␣ during Ovarian Maturation The most significant changes and activation during the vitellogenic process were observed for PKC ␣ (Fig. 1). In stage “A” of ovarian maturation, this isoenzyme (with a molecular mass of approximately 80 kDa) was present only in the cytosolic fraction (Fig. 1, ␣ c). Upon maturation to stage “B”, an increase in the total amount of the isoenzyme was accompanied by translocation from the cytosol to the particulate fraction (Fig. 1, ␣ B m) and the appearance of predominant low- and high-molecular-weight species, primarily in the particulate fraction (presumably membranes; Fig. 1, ␣ B). These processes were augmented during the following maturation stage (Fig. 1, ␣ C), whereas at the end of the maturation process the holoenzyme apparently reappeared in the cytosol, accompanied by an increase in the abundance of the high- and lowmolecular-weight species in the particulate fraction (Fig. 1, ␣ D). Since PKC ␣ changed so dramatically during the vitellogenic process, the onset of PKC ␣ activation was investigated to determine whether this activation coincided with the secondary vitellogenic stage. A model system, the intersex C. quadricarinatus individ-


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FIG. 2. Activation of vitellogenesis and PKC ␣ in ovaries of andrectomized intersex C. quadricarinatus. (A) Polypeptide profile separated on SDS–PAGE and stained with Coomassie blue. (B) Immunoblot of the same gel using anti-PKC ␣. Ovary from an intact intersex individual, with an average oocyte diameter of 319 ␮m, served as control (con). Andrectomized intersex ovary (ag) had an average oocyte diameter of 1360 ␮m. c, cytosol; m, particulate fraction; rb, rat brain cytosol.

ual, in which the ovary is always arrested at the primary vitellogenic stage (Sagi et al., 1996a; Khalaila et al., 1999) was used. Ablation of the androgenic gland of such intersex animals results in instant ovarian maturation, as manifested in an increase of the average oocyte diameter from 320 to 1360 ␮m and a dramatic change in the Coomassie-blue-stained SDS– PAGE polypeptide profile of the ovary (Fig. 2A). In parallel to the onset of secondary vitellogenesis, dramatic activation of PKC ␣ was observed, as was demonstrated by the appearance of high-molecular-weight (⬃120-kDa) PKC in the ovarian particulate fraction (Fig. 2B, ag-). The cytosol and membrane profiles of intact intersex animals contained mainly low-molecular-weight PKC ␣-related polypeptides (Fig. 2B, con), apparently degraded PKC, similar to the rat brain cytosol enzyme profile, which also contained degraded peptides (Fig. 2B, rb). It thus seems that most of the PKC ␣ in the arrested intersex ovaries was degraded, as indicated by the minute amounts of 80kDa “holoenzyme” observable at longer exposure times to the X-ray film in the immunoblot development (not shown).

Changes in Localization of PKC ␣ during Ovarian Maturation To follow the fate and cellular localization of PKC ␣ at different stages of maturation, immunohistological


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staining was performed on thin sections of ovaries (Fig. 3). In Fig. 3A it can be seen that in premature oocytes (oocyte diameter ⬍100 ␮m) PKC ␣ immunoreactivity was lodged mainly in the cytoplasm. As oocyte maturation advanced, PKC ␣ appeared in specific vesicles (Figs. 3B and 3C). Some PKC ␣ immunoreactivity was observed in the cytoplasmic membrane of the oocyte (Figs. 3B and 3C). Upon further maturation, the following changes took place in the localization of PKC ␣. The isoenzyme appeared to be present in the cytoplasm and concentrated around the nuclei and oocyte membranes, especially in the parts surrounding peripheral lipid vesicles. In addition, some PKC ␣ appeared in a diffuse (not vesicular) form in the cytosol (Figs. 3C and 3D). The picture derived from the localization study (Fig. 3) was thus in keeping with the previous finding of PKC ␣ primarily in the cytosol in the premature oocyte and of its translocation to the membranes during maturation and vitellogenesis (Figs. 1 and 2).

Effect of Phorbol Ester and Methyl Farnesoate on the Enzymatic Activity of PKC in the Ovary To verify the immunoblot and immunostain findings, an attempt was made to demonstrate PKC activity in a crude ovarian extract and in ovarian organ culture. In a typical experiment with a crude extract of vitellogenic ovary, histone phosphorylation by

FIG. 3. Immunostaining localization (I) of PKC ␣ in ovaries of C. quadricarinatus at different vitellogenic stages. S, stained with hematoxylin and eosin. (A) Small oocytes in premature ovary (average oocyte diameter 250 ␮m). (B) Primary vitellogenic stage (average oocyte diameter 300 ␮m). (C) Early secondary-vitellogenic ovary (average oocyte diameter of 500 ␮m). (D) Secondary-vitellogenic ovary (average oocyte diameter 1500 ␮m). Bars are expressed in ␮m.

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DISCUSSION

FIG. 4. Activation of PKC in C. quadricarinatus ovarian homogenate by TPA, phosphatidylserine, and methyl farnesoate (MF). A representative case of activation of vitellogenic ovarian homogenate was analyzed for histone phosphorylation and presented as cpm incorporation into histone. CONT, control.

[␥- 32P]ATP was found to be activated by phosphatidylserine plus TPA, a phorbol ester known to be an activator of mammalian PKC (Fig. 4). Similar activation was obtained in a previtellogenic ovarian extract (not shown). To validate the premise that methyl farnesoate participates in ovarian maturation by a direct effect on PKC activity, its ability to serve as an activator in the histone phosphorylation assay was evaluated. Phosphatidylserine-dependent methyl farnesoate activation was indeed found in the in vitro phosphorylation assay (Fig. 4). Total TPA- and methyl farnesoate-dependent phosphatidylserine activity was 35 ⫾ 3% and 27 ⫾ 10% over the control, respectively. Furthermore, when the effects of TPA and methyl farnesoate were studied in organ culture of ovarian fragments, methyl farnesoate activated translocation of PKC ␣ to the particulate fraction, as was shown for TPA (Fig. 5). Moreover, methyl farnesoate and TPA induced the appearance of similar high-molecularweight species of PKC ␣ (⬃120 kDa, Fig. 5). The actual induction of PKC ␣ activity was demonstrated by the appearance of immunoreactive polypeptides to threonine phosphate antibody induced by TPA and methyl farnesoate (⬃40-, ⬃60-, and ⬃120 kDa; Fig. 5, arrows indicate apparent autophosphorylation of PKC ␣ species). Activated protein-bound tyrosine phosphate was not found in this activation system (not shown).

In the present study on C. quadricarinatus, the electrophoretic profiles of various PKC isoenzymes showed differential changes during the vitellogenic process. Some isoenzyme exhibited large quantitative changes, whereas others showed only minor changes. During vitellogenesis, PKC isoenzymes were translocated from the cytosol fraction to the particulate fractions, indicating PKC activation. Activation of the various PKC isoenzymes did not seem to be synchronous, suggesting a different role for each isoenzyme during the maturation process. In this respect, PKC isoenzymes of C. quadricarinatus are similar to mammalian PKC isoenzymes, which have distinct functions during cell differentiation and development, as has been shown in a human promyelocytic leukemia cell line (Hashimoto et al., 1990). PKC has also been found to be involved in gonad and oocyte maturation in mammals before and after fertilization (Steele and Leung, 1992; Gallicano et al., 1997). In the rat, for example, the ratio of PKC isoenzymes in the corpora lutea changed quantitatively during ovarian differentiation (Cutler et al., 1993), and PKC subspecies were expressed in the pituitary gland during maturation (Garcia Navarro et al., 1994). Concomitant with PKC activation, high- and lowmolecular-weight immunoreactive polypeptides appeared during the maturation process in C. quadricarinatus. The low-molecular-weight species were

FIG. 5. Immunoblot profile of PKC ␣ and phosphothreonine residue in polypeptides of the cytosol and particulate fraction of C. quadricarinatus. Induction of translocation by TPA and methyl farnesoate in an early-vitellogenic ovary with an average oocyte diameter of 600 ␮m. c, cytosol; m, particulate fraction; CONT, control. Arrows represent phosphorylated polypeptides that appear both in the methyl farnesoate- and TPA-treated ovaries; these polypeptides show similarities in both PKC ␣ and phosphothreonine profiles.


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probably produced by the proteolytic action of calpain, as was the case for the two major proteolysis products (47- to 49-kDa catalytic fragments and a 36kDa regulatory fragment) found in rat brain cytosol (Mikawa, 1990). The high-molecular-weight species might result from PKC translocation and activation in a manner similar to the assembly of a high-molecularweight complex (⬃120 kDa) of PKC ␣ after activation of ubiquitin in human fibroblasts (Lee et al., 1997) or of a ␮-calpain–PKC complex (with an apparent molecular mass close to 190 kDa) in rabbit skeletal muscle (Savart et al., 1995). The high-molecular-weight form reported in the present study (⬃120 kDa) seemed to be cross-linked to membranal polypeptides; this crosslinking could be a step in PKC degradation, similar to the formation of the PKC complexes described above. The mode by which this PKC is constructed, as well as its content, needs to be further investigated. The similarity of the C. quadricarinatus PKC isoenzymes to mammalian PKC was also expressed in the mode of activation in vitro. Ovarian extracts from C. quadricarinatus were activated in vitro by TPA and phosphatidylserine, as for mammalian PKC (Castagna et al., 1982). Similar activation was also demonstrated by methyl farnesoate. Moreover, the mode of stimulation of PKC ␣ in organ culture was similar for TPA and methyl farnesoate: both compounds induced phosphorylation of threonine residues. The phosphothreonine-containing polypeptides were apparently PKC ␣ subspecies, since their molecular weights correlated with those of the immunoreactive PKC ␣ bands. This finding indicates an autophosphorylation pattern for PKC ␣. The activation of PKC ␣ by methyl farnesoate and the findings that PKC ␣ activation took place in parallel to the secondary-vitellogenic stage (as was demonstrated in the C. quadricarinatus intersex model) suggest that methyl farnesoate is involved in the onset of vitellogenesis through the mediation of PKC. This possibility is supported by a number of studies in different species which demonstrated that methyl farnesoate exerts its effect via PKC. Yamamoto et al. (1997) showed induction of larval metamorphosis in B. amphitrite by methyl farnesoate via PKC activation. Biggers and Laufer (1999) demonstrated mediation of juvenile-hormone-induced settlement and metamorphosis of Capitella larvae by PKC and activation of purified rat brain PKC and a PKC-like enzyme in Capitella larval homogenate by methyl farnesoate.


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Furthermore, the group of Sevala and Davey (1993) reported that juvenile hormone activated PKC in the regulation of vitellogenin uptake into the oocyte of the insect Locusta migratoria (Davey, 1996) by causing a decrease in the volume of follicular cells (Davey et al., 1993). In this context, it may be noted that retinoic acid, which has a structure similar to that of methyl farnesoate, induced PKC ␣ activation in mammalian carcinoma cells (Cho et al., 1997; Desai and Niles, 1997). In the C. quadricarinatus ovary, methyl farnesoate might play a role in the regulation of vitellogenin uptake, mediated by PKC activation, similar to the mode of action suggested for juvenile hormone in insects. The present study has demonstrated immunoreactivity between antibodies raised against mammalian and crustacean PKC isoenzymes. The immunoreactive polypeptides had molecular weights similar to those of mammalian PKC isoenzymes (Croquet et al., 1996). Similar immunoreactivity of some PKC isoenzymes has been shown in yeast, wheat germ, and lobster tail muscle (Kuo et al., 1996). The fact that antibodies against mammalian PKCs were cross-reactive with such a diverse group of organisms and even demonstrated similar modes of action points strongly to the conservation of the PKC sequences in evolution and their importance in living organisms. These results are in keeping with previous studies showing conserved sequence in the PKC gene of a broad range of organisms (e.g., Morawetz et al., 1996; Kofler et al., 1998; Mellor and Parker, 1998).

ACKNOWLEDGMENTS We thank Ms. Tikva Zino for maintaining the animals. We also thank Ms. Inez Mureinik for styling the manuscript. A.S. is the incumbent of the Judith and Murray Shusterman Chair for Career Development in Physiology.

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